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Mechanism of entry of human rhinovirus 2 into HeLa cells
VIROLOGY
158, 255-258 (1987)
Mechanism
of Entry of Human Rhinovirus
2 into HeLa Cells
CHRISTOPH NEUBAUER, LELA FRASEL, ERNST KUECHLER, AND DIETER BLWS’ lnstitut fiir Biochemie, Received November
Wahringerstrasse
17, 1090 Vienna, Austria
19, 1986; accepted January 29, 1987
Internalized human rhinovirus 2 (HRVZ) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20” but not at 34” the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20” prior to addition of monensin permits virus synthesis to occur, depending on the 0 1987 Academic Press, Inc. time of preincubation.
alyzed on polyacrylamide gels in the presence of SDS. From these experiments it was established that MAb 2G2 exclusively bound to pH 5-treated virus but did not recognize native virus. MAb 3F8 bound only to native HRV2. MAb 8F5 bound both native as well as acidtreated virions. Polyclonal antiserum was obtained from rabbits immunized with purified HRV2 and was found to react with native as well as with acid-treated virus to the same extent. In order to test the influence of the acidic environment of the intracellular vesicles on the viral conformation, HeLa cells were incubated with 35S-labeled HRV2 at 34” either in the absence (Fig. 1A) or in the presence of 2 PM monensin (Fig. 1 B). Samples were removed immediately after addition of virus (0 min) and after 30 min and the cells were pelleted by a short centrifugation. The cellular supernatants and the cell pellets were processed separately for immunoprecipitation with 2G2. Viral material not bound by 2G2 was subsequently precipitated with polyclonal antiserum. Both precipitates were analyzed on 12.5% polyacrylamide gels in the presence of SDS. Immediately after addition of the virus to the cell suspension 2G2 failed to precipitate any viral material from the cell supernatants (lane 1) or from the solubilized cell pellets (lane 3) irrespective of the presence of monensin (compare Figs. 1A and 1 B). Despite the short time of contact of the virus with the cells resulting from handling the samples, a rather high amount of virus was recovered from the cell pellets with polyclonal antiserum (lane 4). This is in accordance with the observation of a very rapid binding of HRV2 to HeLa cells at 34” (14, 15). Virus still in the supernatants was precipitated with polyclonal antiserum (lane 2). After 30 min incubation at 34” viral material recognized by 2G2 was present in the cell supernatant as well as in the cell pellet only in the absence of mo-
Binding of rhinoviruses to the cell and subsequent internalization leads to a conformational change (eclipse) giving rise to subviral particles which exhibit a new set of antigenic determinants (C-determinants) not present on native virions (l-3). Similar particles can also be obtained in vitro upon incubation of virus at pH 5 (0. For enveloped viruses, it has been shown that receptor-mediated internalization delivers the virus-receptor complex to endosomes, vesicles which are acidified to a pH of 5-O-5.5 (4). This acidic environment then triggers a conformational change leading to viral entry into the cytoplasm (5-7). Recently receptor-mediated endocytosis has also been proposed as a mode of entry of picornaviruses (8-7 7). Substances known to increase the pH of intracellular vesicles such as NH&I, monensin, and others were shown to protect HeLa cells against infection by poliovirus and by human rhinovirus 2 (HRV2) (9). The present study was initiated to gain more insight into the infection pathway of rhinoviruses. The results obtained point to the endosomal compartment as being the site of uncoating. Monoclonal antibodies (MAbs) were generated using spleen cells obtained from mice immunized with purified HRV2 following published protocols (12). The screening of the hybridomas lead to the discovery of one clone (2G2) which showed a strong reaction in the ELISA but which was negative in a microneutralization test. Two others, both producing neutralizing antibodies, showed either weak (3F8) or strong (8F5, see Ref. (13)) reaction in the ELISA. Staphylococcus aureusaided immunoprecipitation of either native or pH 5treated (1, 3) [35S]methionine-labeled HRV2 was carried out employing ascites fluids obtained from the cloned hybridomas. The immunoprecipitated material was an’ To whom requests for reprints should be addressed.
255
0042.6822/87 $3.00 Copyright 0 1997 by Academic Press, Inc. All rights of reproduction I” any form resewed.
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FIG. 1. Effect of incubation of 36S-labeled HRV2 with HeLa cells at 34’ in the absence (A) and in the presence of monensin (B). Growth of HRV2 in HeLa cell suspension culture and purificiation of virus were done essentially as described previously (22). For immunoprecipitation MAbs were bound to Staphy/ococcus aureus cells (BRL) via rabbit anti-mouse IgG (Bio Yeda). IgG of polyclonal rabbit antiserum was directly bound to the S. aureus cells. HeLa cells (5 X 1 OS)were suspended in 0.5 ml of minimum essential medium (GIBCO) containing 2% heat-inactivated fetal calf serum and 30 mM MgC12 in Eppendorf vials (without monensin (A), with 2 pM monensin (B)). [36SlMethionine (Amersham)-labeled HRV2 (1 O4cpm) (75) (centrifuged for 5 min in an Eppendorf centrifuge) was added and the vials were slowly rotated in a water bath at the specified temperatures. The cells were pelleted, the supernatant was saved for immunoprecipitation, and the cell pellet was suspended in 0.15 ml RIPA buffer (23) for 20 min at 0”. Cell debris was removed by centrifugation. The supernatants were incubated with 20 pi of the immunoadsorbents for 1 hr at room temperature and the bacterial cells were pelleted, washed twice with RIPA buffer and twice with PBS, boiled in Laemmli sample buffer (24) and analyzed on 12.5% polyacrylamide gels in the presence of SDS (24). This gel system resolves only VP1 , VP2, and VP3. The gels were soaked in 1 M sodium salicylate for 30 min, dried, and exposed to X-ray film. lmmunoprecipitations were carried out with MAb 2G2 from the cell supernatant at 0 min, lane 1, and after 30 min, lane 5; from the solubilized cell pellets at 0 min, lane 3, and after 30 min, lane 7; material not precipitated by 2G2 was subsequently precipitated with polyclonal antiserum from the cell supernatants at 0 min, lane 2, and after 30 min, lane 6; from the solubilized cell pellets at 0 min, lane 4, and after 30 min, lane 8.
nensin (compare Figs. 1A and 1B, lanes 5 and lanes 7, respectively). It is evident that the virus recovered with 2G2 from the cell pellet after 30 min of incubation in absence of monensin has undergone extensive degradation (Fig. 1A, lane 7). VP1 was cleaved and VP2 had mostly disappeared. The structure of the antigenic determinant recognized by 2G2 was apparently not modified by this proteolysis as these subviral particles were still precipitated. In the presence of monensin no viral material was precipitated by 2G2 either in the supernatant (Fig. 1 B, lane 5) or in the cell pellet (lane 7). The virus was, however, bound by the polyclonal antiserum (lane 8). Furthermore proteolysis does not occur in presence of monensin. Control experiments employing proteinase K digestion of the cell surface (76) demonstrated that about 70% of cell associated virus
was internalized after 30 min at 34” in the absence as well as in the presence of 2 pM monensin when compared to a background of 18% obtained with formaldehyde-fixed cells (data not shown). For a variety of different cell types it has been shown that fusion of endosomes with lysosomes is strongly inhibited at 20” (5, 7, 17-19). Hence the virions are expected to accumulate in the prelysosomal compartment and proteolysis should be prevented at this temperature. The incubation periods were extended up to 120 min because of the lower efficiency of the attachment process at 20’ as compared to 34” (I, 15). Material immunoprecipitated from the solubilized cell pellets is shown in Fig. 2. A continuous increase of virus particles in the C-conformation can clearly be seen (lanes 3, 5, 7, and 9 corresponding to 15, 30, 60, and 120 min, respectively). Subsequent precipitation employing polyclonal antiserum (lanes 4, 6, 8, and 10) showed a concomitant decrease of bound viral material as expected. At this temperature no detectable binding to the cells has occurred at 0 min (lanes 1 and 2). After 2 hr of incubation all the virions exhibited the C-determinant (compare lanes 9 and 10); however, no proteolytic degradation could be observed. The following experiments were designed to investigate the significance of the modification of the viral capsid in viva for the infectious pathway, taking into account the localization of the virus in different cellular compartments at 20” vs 34”. HeLa cells (2 X 105) were incubated with 2 pM monensin in 0.4 ml methioninefree medium for 30 min before challenge with HRV2 at an m.o.i. of 300. After an attachment period of 20 min at 34” the cells were washed and the incubation was continued for 4 hr in fresh methionine-free medium 0
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tIllI”.
VP1 -
--llr
VP2VP3’
-,rr*c--*reamwmqw-*
12345678910 FIG. 2. Effect of incubation of 35S-labeled HRV2 with HeLa cells at 20”. lmmunoprecipitation were carried out from the solubilized cell pellets with MAb 2G2 (as described in Fig. 1) at 0 min, lane 1; after 15 min, lane 3; 30 min, lane 5; 60 min, lane 7; 120 min, lane 9. lmmunoprecipitations were performed subsequently with polyclonal antiserum at 0 min, lane 2; 15 min, lane 4; 30 min, lane 6; 60 min, lane 8; 120 min, lane 10.
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containing 2 PM monensin. [35S]Methionine (20 &i) was then added and after an additional 12 hr at 34” the ceils were lysed with RIPA buffer. Under these conditions only one viral growth cycle takes place (20). Cell debris was removed by a low-speed centrifugation and virus progeny was precipitated from the supernatant with a mixture of MAbs 3F8 and 8F5 bound to S. aureus cells. In a parallel control experiment monensin was omitted throughout the whole incubation period. The precipitates were analyzed on a polyacrylamide gel (Fig. 3). In the absence of monensin, VPl, VP2, and VP3 as well as VP0 (the precursor of VP4 and VP2) were found to be labeled (lane 1). If, however, monensin was present throughout the infection cycle, viral synthesis did not occur (lane 2). If the block of viral infection by monensin were due to the lack of vesicle acidification, it should be possible to overcome this inhibition by artificially decreasing the pH of the intracellular vesicles (21). When cells in the presence of 2 pM monensin were subjected to an incubation with pH 5.5 buffer (30 mM sodium acetate, 10 mM sodium phosphate, 5 mM KCI, 110 mM NaCI) for 20 min immediately after viral infection at 34”, viral synthesis was completely restored (compare lane 3 to lane 1). If, however, the infected cells were incubated for 2 hr at 34” before treatment with the pH 5.5 buffer, no virus was produced (lane 4). Viral attachment (for
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123466 FIG. 3. Influence of monensin on virus synthesis at different temperatures Cells were infected with HRV2 and incubated for 20 min at 34”, lane 1; for 20 min at 34” in the presence of 2 pM monensin, lane 2; for 20 min at 34” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 3; for 2 hr at 34” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 4; for 30 min at 20” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 5; for 2 hr at 20” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 6. Cells were pelleted, resuspended in methionine-free medium containing 2% dialyzed fetal calf serum, and further incubated at 34”. In addition, 2 pM monensin was added to all samples except to the control (lane 1). After 4 hr [%S]methionine was added and the incubation was continued for 12 hr. lmmunoprecipitation was carried out with a mixture of MAbs 3F8 and 8F5 which resulted in somewhat reduced background as compared to polyclonal antiserum (not shown).
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30 min) and the incubations were then carried out at 20”. After the treatment with pH 5.5 buffer the temperature was, however, shifted to 34” to allow protein synthesis to proceed at a normal rate. The cells were washed and subjected either immediately (lane 5) or after a 2-hr incubation at 20” to the treatment with pH 5.5 buffer for 40 min (lane 6). In contrast to the incubations at 34” (lanes 3 and 4) viral synthesis could be restored by the pH 5.5 treatment even at 2 hr after inoculation (lanes 5 and 6). This indeed suggests the transport of the virus through different cellular compartments which have distinct capabilities to initiate uncoating upon acidification. Efficient uncoating would thus be expected to be possible even at 20” provided that the natural acidification of the vesicles was not inhibited. Cells (2 X 105) were incubated with virus at an m.o.i. of 300. At different time periods monensin was added and was present throughout the following incubations. The cells were washed and shifted to 34” and after an additional 4 hr, 20 &i [35S]methionine was added. Twelve hours after viral infection cells were lysed and processed for gel electrophoresis (Fig. 4). If monensin was added before virus addition no viral proteins could be detected (lane 1). However, incubation of the inoculated cells at 20” prior to the addition of monensin permitted viral synthesis to occur in a time-dependent manner (lanes 2-5, at 30 min, 1, 2,4 hr of preincubation, respectively). Our results show that productive infection in the presence of monensin can be brought about by artificial acidification provided that the HRV2 has previously entered the appropriate cellular compartment. At 34” viral infectivity can be rescued only if the pH 5.5 treatment is carried out shortly after inoculation but it is readily lost after 2 hr of incubation (Fig. 3, lane 3 compared to lane 4). At 20”, however, infectivity can be recovered even after 2 hr (Fig. 3, lane 5 compared to lane 6). These findings suggest a pathway of the internalized virus through different cellular compartments. The progression from one (probably endosomes) to the other (probably lysosomes) is however blocked at 20”. At 34” the virus which failed to be uncoated apparently rapidly proceeds to secondary lysosomes where it aborts. It is therefore unlikely that HRV2 enters the cytoplasm from lysosomes to any large extent. Almost all of the input virus exhibits the C-conformation when the infected cells are incubated at 20” for 1 hr (Fig. 2, lanes 7 and 8). Nevertheless the efficiency of infection still increases after 1 hr of incubation at 20” (Fig. 4). The structural modification of the viral capsid to the C-conformation is therefore clearly not the rate-limiting step in infection. The correlation between the incubation time at 20” and the virus yield obtained suggests that the endosomal compartment
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VP1 vpo= VP2, VP3’
12345 FIG. 4. Influence of the time of addition of monensin on virus synthesis at 20’. Cells were infected with HRV2 and newly synthesized viral proteins were labeled with [%lmethionine. Incubation was carried out at 20’ and monensin was added at 0 min, lane 1; after 30 min, lane 2; 1 hr, lane 3; 2 hr, lane 4; 4 hr, lane 5. After an additional 30 min at 20’ cells were pelleted, resuspended in methionine-free medium, 296 dialyzed fetal calf serum, and 2 PM monensin, and further incubated at 34”. After 4 hr [%]methionine was added and the incubation was continued for 12 hr. lmmunoprecipitation was performed with a mixture of MAbs 3F8 and 8F5.
mediates an essential step in infection (Fig. 4). The results support the notion that penetration of HRV2 into the cytoplasm and/or uncoating takes place primarily from endosomes. ACKNOWLEDGMENTS This work was supported by the “&terreichischer Fonds zur Forderung der Wissenschaftlichen Forschung.” We are indebted to T. Skern and Z. Rattler for valuable discussions and to T. Lenert for typing the manuscript.
REFERENCES 7. KORANT, B. D., LONBERG-HOLM, K., NOBLE, J., and STASNY, J. T., Virology 48, 7 l-86 (1972).
2. KORANT,B. D., LONBERG-HOLM,K.. YIN, F. H., and NOBLE-HARVEY, J., virology 63, 384-394 (1975). 3. LONBERG-HOLM,K., and YIN, F. H., J. viral. 12, 114-l 23 (1973). 4. WILEMAN, TH., HARDING, C., and STAHL, PH., Biochem. 1. 232, l14 (1985). 5. WHITE, J., and HELENIUS,A., Proc. Nat/. Acad. Sci, USA 77, 32733277 (1980). 6. MARSH, M., BOLZAU, E., and HELENIUS, A., Cell 32, 931-940 (1983). 7. YOSHIMURA,A., and OHNISHI, %-I., J. Viral. 51, 497-504 (1984). 8. MADSHUS, I. H., OLSNES, S., and SANDVIG, K., J. Cell Biol. 98, 1194-1200(1984). 9. MADSHUS, I. H., OLSNES,S.. and SANDVIG,K., virology 139, 346357 (1984). 10. CARILLO,E. C., GIACHETTI,C., and CAMPOS, R., !Iiro/ogy 147, 1 18125 (1985). 11. ZEICHHARDT, H., WETZ, K., WILLINGMANN, P., and HABERMEHL, K.-O., J. Gen. Virol. 66, 483-492 (1985). 12. GALFR~, G., and MILSTEIN, C., In “Methods in Enzymology” (J. 1. Langone and H. van Vunakis, Eds.), Vol. 73, pp. 3-46. Academic Press, New York, 1982. 13. SKERN, T.. NEUBAUER,CH., FRASEL, L., GRUENDLER,P., SOMMERGRUBER,W., ZORN, M., KUECHLER,E., and BLAAS, D., J. Gen. Viral. 68, 315-323 (1987). 14. LONBERG-HOLM,K., and KORANT, B. D., 1. Viral. 9, 29-40 (1972). 15. LONBERG-HOLM, K., and WHITELEY, N. M., J. Viral. 19, 857-870 (1976). 16. HELENIUS, A., KARTENBECK,J., SIMONS, K., and FRIES, E., 1. Cell Biol. 84, 404-420 (1980). 17. DUNN, W. A., HUBBARD,A. L., and ARONSON, N. N., 1. Biol. Chem. 255,5971-5978 (1980). J., and HOP18. MILLER, K., BEARDMORE,J., KANETY.H., SCHLESSINGER, KINS, C. R., J. Cell Biol. 102, 500-509 (1986). 19. RICHMAN, D. D., HOSTETLER,K. Y., YAZAKI, P. J., and CLARK, S., Virology 151,200-216 (1986). 20. THOMAS, D. C., CONANT, R. M., and HAMPARIAN,V. V., Proc. Sot. fxp. Biol. Med. 133, 62-65 (1970). 21. SANDVIG, K., and OLSNES, S., 1. Cell Biol. 87, 828-832 (1980). 22. SKERN, T., SOMMERGRUBER,W., Bvv\s, D., PIELER, CH., and KUECHLER,E., Virology 136, 125-l 32 (1984). 23. COLLET, M., and ERIKSON,R. L.. Proc. Nat/, Acad. Sci. USA 74, 2021-2024 (1978). 24. LAEMMLI, U. K., Nature (London) 227, 680-685 (1970).
158, 255-258 (1987)
Mechanism
of Entry of Human Rhinovirus
2 into HeLa Cells
CHRISTOPH NEUBAUER, LELA FRASEL, ERNST KUECHLER, AND DIETER BLWS’ lnstitut fiir Biochemie, Received November
Wahringerstrasse
17, 1090 Vienna, Austria
19, 1986; accepted January 29, 1987
Internalized human rhinovirus 2 (HRVZ) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20” but not at 34” the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20” prior to addition of monensin permits virus synthesis to occur, depending on the 0 1987 Academic Press, Inc. time of preincubation.
alyzed on polyacrylamide gels in the presence of SDS. From these experiments it was established that MAb 2G2 exclusively bound to pH 5-treated virus but did not recognize native virus. MAb 3F8 bound only to native HRV2. MAb 8F5 bound both native as well as acidtreated virions. Polyclonal antiserum was obtained from rabbits immunized with purified HRV2 and was found to react with native as well as with acid-treated virus to the same extent. In order to test the influence of the acidic environment of the intracellular vesicles on the viral conformation, HeLa cells were incubated with 35S-labeled HRV2 at 34” either in the absence (Fig. 1A) or in the presence of 2 PM monensin (Fig. 1 B). Samples were removed immediately after addition of virus (0 min) and after 30 min and the cells were pelleted by a short centrifugation. The cellular supernatants and the cell pellets were processed separately for immunoprecipitation with 2G2. Viral material not bound by 2G2 was subsequently precipitated with polyclonal antiserum. Both precipitates were analyzed on 12.5% polyacrylamide gels in the presence of SDS. Immediately after addition of the virus to the cell suspension 2G2 failed to precipitate any viral material from the cell supernatants (lane 1) or from the solubilized cell pellets (lane 3) irrespective of the presence of monensin (compare Figs. 1A and 1 B). Despite the short time of contact of the virus with the cells resulting from handling the samples, a rather high amount of virus was recovered from the cell pellets with polyclonal antiserum (lane 4). This is in accordance with the observation of a very rapid binding of HRV2 to HeLa cells at 34” (14, 15). Virus still in the supernatants was precipitated with polyclonal antiserum (lane 2). After 30 min incubation at 34” viral material recognized by 2G2 was present in the cell supernatant as well as in the cell pellet only in the absence of mo-
Binding of rhinoviruses to the cell and subsequent internalization leads to a conformational change (eclipse) giving rise to subviral particles which exhibit a new set of antigenic determinants (C-determinants) not present on native virions (l-3). Similar particles can also be obtained in vitro upon incubation of virus at pH 5 (0. For enveloped viruses, it has been shown that receptor-mediated internalization delivers the virus-receptor complex to endosomes, vesicles which are acidified to a pH of 5-O-5.5 (4). This acidic environment then triggers a conformational change leading to viral entry into the cytoplasm (5-7). Recently receptor-mediated endocytosis has also been proposed as a mode of entry of picornaviruses (8-7 7). Substances known to increase the pH of intracellular vesicles such as NH&I, monensin, and others were shown to protect HeLa cells against infection by poliovirus and by human rhinovirus 2 (HRV2) (9). The present study was initiated to gain more insight into the infection pathway of rhinoviruses. The results obtained point to the endosomal compartment as being the site of uncoating. Monoclonal antibodies (MAbs) were generated using spleen cells obtained from mice immunized with purified HRV2 following published protocols (12). The screening of the hybridomas lead to the discovery of one clone (2G2) which showed a strong reaction in the ELISA but which was negative in a microneutralization test. Two others, both producing neutralizing antibodies, showed either weak (3F8) or strong (8F5, see Ref. (13)) reaction in the ELISA. Staphylococcus aureusaided immunoprecipitation of either native or pH 5treated (1, 3) [35S]methionine-labeled HRV2 was carried out employing ascites fluids obtained from the cloned hybridomas. The immunoprecipitated material was an’ To whom requests for reprints should be addressed.
255
0042.6822/87 $3.00 Copyright 0 1997 by Academic Press, Inc. All rights of reproduction I” any form resewed.
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FIG. 1. Effect of incubation of 36S-labeled HRV2 with HeLa cells at 34’ in the absence (A) and in the presence of monensin (B). Growth of HRV2 in HeLa cell suspension culture and purificiation of virus were done essentially as described previously (22). For immunoprecipitation MAbs were bound to Staphy/ococcus aureus cells (BRL) via rabbit anti-mouse IgG (Bio Yeda). IgG of polyclonal rabbit antiserum was directly bound to the S. aureus cells. HeLa cells (5 X 1 OS)were suspended in 0.5 ml of minimum essential medium (GIBCO) containing 2% heat-inactivated fetal calf serum and 30 mM MgC12 in Eppendorf vials (without monensin (A), with 2 pM monensin (B)). [36SlMethionine (Amersham)-labeled HRV2 (1 O4cpm) (75) (centrifuged for 5 min in an Eppendorf centrifuge) was added and the vials were slowly rotated in a water bath at the specified temperatures. The cells were pelleted, the supernatant was saved for immunoprecipitation, and the cell pellet was suspended in 0.15 ml RIPA buffer (23) for 20 min at 0”. Cell debris was removed by centrifugation. The supernatants were incubated with 20 pi of the immunoadsorbents for 1 hr at room temperature and the bacterial cells were pelleted, washed twice with RIPA buffer and twice with PBS, boiled in Laemmli sample buffer (24) and analyzed on 12.5% polyacrylamide gels in the presence of SDS (24). This gel system resolves only VP1 , VP2, and VP3. The gels were soaked in 1 M sodium salicylate for 30 min, dried, and exposed to X-ray film. lmmunoprecipitations were carried out with MAb 2G2 from the cell supernatant at 0 min, lane 1, and after 30 min, lane 5; from the solubilized cell pellets at 0 min, lane 3, and after 30 min, lane 7; material not precipitated by 2G2 was subsequently precipitated with polyclonal antiserum from the cell supernatants at 0 min, lane 2, and after 30 min, lane 6; from the solubilized cell pellets at 0 min, lane 4, and after 30 min, lane 8.
nensin (compare Figs. 1A and 1B, lanes 5 and lanes 7, respectively). It is evident that the virus recovered with 2G2 from the cell pellet after 30 min of incubation in absence of monensin has undergone extensive degradation (Fig. 1A, lane 7). VP1 was cleaved and VP2 had mostly disappeared. The structure of the antigenic determinant recognized by 2G2 was apparently not modified by this proteolysis as these subviral particles were still precipitated. In the presence of monensin no viral material was precipitated by 2G2 either in the supernatant (Fig. 1 B, lane 5) or in the cell pellet (lane 7). The virus was, however, bound by the polyclonal antiserum (lane 8). Furthermore proteolysis does not occur in presence of monensin. Control experiments employing proteinase K digestion of the cell surface (76) demonstrated that about 70% of cell associated virus
was internalized after 30 min at 34” in the absence as well as in the presence of 2 pM monensin when compared to a background of 18% obtained with formaldehyde-fixed cells (data not shown). For a variety of different cell types it has been shown that fusion of endosomes with lysosomes is strongly inhibited at 20” (5, 7, 17-19). Hence the virions are expected to accumulate in the prelysosomal compartment and proteolysis should be prevented at this temperature. The incubation periods were extended up to 120 min because of the lower efficiency of the attachment process at 20’ as compared to 34” (I, 15). Material immunoprecipitated from the solubilized cell pellets is shown in Fig. 2. A continuous increase of virus particles in the C-conformation can clearly be seen (lanes 3, 5, 7, and 9 corresponding to 15, 30, 60, and 120 min, respectively). Subsequent precipitation employing polyclonal antiserum (lanes 4, 6, 8, and 10) showed a concomitant decrease of bound viral material as expected. At this temperature no detectable binding to the cells has occurred at 0 min (lanes 1 and 2). After 2 hr of incubation all the virions exhibited the C-determinant (compare lanes 9 and 10); however, no proteolytic degradation could be observed. The following experiments were designed to investigate the significance of the modification of the viral capsid in viva for the infectious pathway, taking into account the localization of the virus in different cellular compartments at 20” vs 34”. HeLa cells (2 X 105) were incubated with 2 pM monensin in 0.4 ml methioninefree medium for 30 min before challenge with HRV2 at an m.o.i. of 300. After an attachment period of 20 min at 34” the cells were washed and the incubation was continued for 4 hr in fresh methionine-free medium 0
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12345678910 FIG. 2. Effect of incubation of 35S-labeled HRV2 with HeLa cells at 20”. lmmunoprecipitation were carried out from the solubilized cell pellets with MAb 2G2 (as described in Fig. 1) at 0 min, lane 1; after 15 min, lane 3; 30 min, lane 5; 60 min, lane 7; 120 min, lane 9. lmmunoprecipitations were performed subsequently with polyclonal antiserum at 0 min, lane 2; 15 min, lane 4; 30 min, lane 6; 60 min, lane 8; 120 min, lane 10.
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containing 2 PM monensin. [35S]Methionine (20 &i) was then added and after an additional 12 hr at 34” the ceils were lysed with RIPA buffer. Under these conditions only one viral growth cycle takes place (20). Cell debris was removed by a low-speed centrifugation and virus progeny was precipitated from the supernatant with a mixture of MAbs 3F8 and 8F5 bound to S. aureus cells. In a parallel control experiment monensin was omitted throughout the whole incubation period. The precipitates were analyzed on a polyacrylamide gel (Fig. 3). In the absence of monensin, VPl, VP2, and VP3 as well as VP0 (the precursor of VP4 and VP2) were found to be labeled (lane 1). If, however, monensin was present throughout the infection cycle, viral synthesis did not occur (lane 2). If the block of viral infection by monensin were due to the lack of vesicle acidification, it should be possible to overcome this inhibition by artificially decreasing the pH of the intracellular vesicles (21). When cells in the presence of 2 pM monensin were subjected to an incubation with pH 5.5 buffer (30 mM sodium acetate, 10 mM sodium phosphate, 5 mM KCI, 110 mM NaCI) for 20 min immediately after viral infection at 34”, viral synthesis was completely restored (compare lane 3 to lane 1). If, however, the infected cells were incubated for 2 hr at 34” before treatment with the pH 5.5 buffer, no virus was produced (lane 4). Viral attachment (for
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123466 FIG. 3. Influence of monensin on virus synthesis at different temperatures Cells were infected with HRV2 and incubated for 20 min at 34”, lane 1; for 20 min at 34” in the presence of 2 pM monensin, lane 2; for 20 min at 34” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 3; for 2 hr at 34” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 4; for 30 min at 20” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 5; for 2 hr at 20” in the presence of monensin followed by treatment with pH 5.5 buffer, lane 6. Cells were pelleted, resuspended in methionine-free medium containing 2% dialyzed fetal calf serum, and further incubated at 34”. In addition, 2 pM monensin was added to all samples except to the control (lane 1). After 4 hr [%S]methionine was added and the incubation was continued for 12 hr. lmmunoprecipitation was carried out with a mixture of MAbs 3F8 and 8F5 which resulted in somewhat reduced background as compared to polyclonal antiserum (not shown).
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30 min) and the incubations were then carried out at 20”. After the treatment with pH 5.5 buffer the temperature was, however, shifted to 34” to allow protein synthesis to proceed at a normal rate. The cells were washed and subjected either immediately (lane 5) or after a 2-hr incubation at 20” to the treatment with pH 5.5 buffer for 40 min (lane 6). In contrast to the incubations at 34” (lanes 3 and 4) viral synthesis could be restored by the pH 5.5 treatment even at 2 hr after inoculation (lanes 5 and 6). This indeed suggests the transport of the virus through different cellular compartments which have distinct capabilities to initiate uncoating upon acidification. Efficient uncoating would thus be expected to be possible even at 20” provided that the natural acidification of the vesicles was not inhibited. Cells (2 X 105) were incubated with virus at an m.o.i. of 300. At different time periods monensin was added and was present throughout the following incubations. The cells were washed and shifted to 34” and after an additional 4 hr, 20 &i [35S]methionine was added. Twelve hours after viral infection cells were lysed and processed for gel electrophoresis (Fig. 4). If monensin was added before virus addition no viral proteins could be detected (lane 1). However, incubation of the inoculated cells at 20” prior to the addition of monensin permitted viral synthesis to occur in a time-dependent manner (lanes 2-5, at 30 min, 1, 2,4 hr of preincubation, respectively). Our results show that productive infection in the presence of monensin can be brought about by artificial acidification provided that the HRV2 has previously entered the appropriate cellular compartment. At 34” viral infectivity can be rescued only if the pH 5.5 treatment is carried out shortly after inoculation but it is readily lost after 2 hr of incubation (Fig. 3, lane 3 compared to lane 4). At 20”, however, infectivity can be recovered even after 2 hr (Fig. 3, lane 5 compared to lane 6). These findings suggest a pathway of the internalized virus through different cellular compartments. The progression from one (probably endosomes) to the other (probably lysosomes) is however blocked at 20”. At 34” the virus which failed to be uncoated apparently rapidly proceeds to secondary lysosomes where it aborts. It is therefore unlikely that HRV2 enters the cytoplasm from lysosomes to any large extent. Almost all of the input virus exhibits the C-conformation when the infected cells are incubated at 20” for 1 hr (Fig. 2, lanes 7 and 8). Nevertheless the efficiency of infection still increases after 1 hr of incubation at 20” (Fig. 4). The structural modification of the viral capsid to the C-conformation is therefore clearly not the rate-limiting step in infection. The correlation between the incubation time at 20” and the virus yield obtained suggests that the endosomal compartment
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SHORT COMMUNICATIONS
VP1 vpo= VP2, VP3’
12345 FIG. 4. Influence of the time of addition of monensin on virus synthesis at 20’. Cells were infected with HRV2 and newly synthesized viral proteins were labeled with [%lmethionine. Incubation was carried out at 20’ and monensin was added at 0 min, lane 1; after 30 min, lane 2; 1 hr, lane 3; 2 hr, lane 4; 4 hr, lane 5. After an additional 30 min at 20’ cells were pelleted, resuspended in methionine-free medium, 296 dialyzed fetal calf serum, and 2 PM monensin, and further incubated at 34”. After 4 hr [%]methionine was added and the incubation was continued for 12 hr. lmmunoprecipitation was performed with a mixture of MAbs 3F8 and 8F5.
mediates an essential step in infection (Fig. 4). The results support the notion that penetration of HRV2 into the cytoplasm and/or uncoating takes place primarily from endosomes. ACKNOWLEDGMENTS This work was supported by the “&terreichischer Fonds zur Forderung der Wissenschaftlichen Forschung.” We are indebted to T. Skern and Z. Rattler for valuable discussions and to T. Lenert for typing the manuscript.
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